Methods for screening compounds for estrogenic activity

ABSTRACT

The present invention provides novel assay methods for identifying compounds that may have both estrogen agonist and antagonist properties. In particular, the assay use cells comprising promoters having an AP1 site linked to a reporter gene. Compounds capable of inducing or blocking expression of the reporter gene can thus be identified. The compounds may be further tested for the ability to modulate the standard estrogen response, as well.

This is a continuation in part of U.S. Ser. No. 08/115,161 filed Sep. 1,1993, now abandoned, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Many breast tumors require estrogens for tumor growth. Thus, treatmentwith antiestrogen compounds can slow or prevent tumor spread. Manyantiestrogens, however, show both estrogen antagonistic and agonisticactivity. The nonsteroidal antiestrogen tamoxifen, for example, which isestablished as the treatment of choice for the endocrine therapy ofadvanced breast cancer, shows both agonistic and antagonistic activity.Sutherland, S. & Jackson, M. Cancer Treat. Revs. 15:183-194 (1987).

The agonistic activity of tamoxifen and other antiestrogens may haveprofound effects upon patients. For example, agonistic activity may havebeneficial effects, such as preventing osteoporosis and reducing serumcholesterol. Love, et al. New Eng. J. Med. 326:852-856 (1992). Love, etal. J. Natl. Cancer Inst. 82:1327-1332 (1990). Conversely, agonisticactivity may also be harmful. Tamoxifen for example sometimes increasesendometrial tumor incidence Iino, et al. Cancer Treat. & Res. 53:228-237 (1991) or switches from inhibition to stimulation of estrogendependent growth in breast tumor progression. Parker, M. G. (ed) CancerSurveys 14: Growth Regulation by Nuclear Hormone Receptors. Cold SpringHarbor Laboratory Press (1992).

It is desirable to identify pure antiestrogens as they are anticipatedto provide more rapid, complete or longer-lasting tumor responses.Wakeling, A. E. Breast Cancer Res. & Treat. 25:1-9 (1993). For example,ICI 164,384 (hereinafter referred to as "ICI"), thought to be a pureantiestrogen, blocked MCF-7 cell invasion activity of a re-constitutedbasement membrane while estradiol and 4'-hydroxytamoxifen stimulatedthis activity suggesting that early treatment of breast cancer with apure antiestrogen might be particularly beneficial in limiting tumorspread. Braacke, et al., Br. J. Cancer 63:867-872 (1991).

Conversely, while pure antiestrogens appear preferable for cancertreatments, mixed agonist-antagonist compounds may be preferable forpreventative treatment. Such compounds should combine sufficientantagonist activity on estrogen stimulated breast tumor growth whilemaintaining simultaneous agonist activity on bone density and serumlipid levels.

In addition, a number of non-steroidal natural and synthetic compoundsfound in the environment have been shown to possess estrogenic activity.For instance, plant flavonoids including genistein and coumestrol andsynthetic compounds such as phenolphthalein, alkylphenols, anddihydroxystilbenes, have been shown to be agonists of the estrogenreceptor (Miksicek, Mol. Pharmacol. 44:37-43 (1993); Nieto et al.,Biochem. Int. 21:305-311 (1990); White et al., Endocrinology 135:175-182(1994); Makela et al., Environ. Health Perspect. 102:572-578 (1994);Krishnan et al. Endocrinology 132:2279-2286 (1993)).

Environmental estrogens, or xenoestrogens, are suspected of playing arole in the causation of a number of diseases such as breast and othercancers. In addition, such compounds may be implicated in humaninfertility and problems in wildlife reproduction. In the case of breastand other cancers, established risk factors (e.g., genetic factors) donot always account for the high levels of of these diseases. Evidencesuggests that lifetime exposure to various xenoestrogens may beimportant in the induction of breast cancer. To the extent suchxenoestrogens are important in diseases such as breast cancer, reductionin exposure to these compounds should be critical to reducing cancerrisks (Davis et al. Environmental Health Perspectives 101:372-377(1993)).

Currently, antiestrogen compounds or xenoestrogenic compounds arescreened with animal models such as the rat uterine test. These testsare cumbersome, slow, expensive and of uncertain application to humansbecause of differences between the human and rodent estrogen receptors.

The prior art fails to provide methods for quickly and easily testingpotential antiestrogen compounds for agnostic as well as antagonisticproperties mediated through pathways other than the classical estrogenresponse pathway, that may affect, adversely or beneficially, their usein various therapeutic applications. In addition, the ability to quicklyand inexpensively screen environmental compounds for estrogenic activityis particularly important for assessing health consequences of new andexisting chemicals. This invention addresses these and other problems inthe art.

SUMMARY OF THE INVENTION

The present invention provides methods for screening test compounds, forexample environmental compounds, for the ability to activate or inhibittranscription through an indirect estrogen response or classicalestrogen response. The indirect estrogen response is mediated bypromoters comprising an AP1 site and the classical estrogen response ismediated by promoters comprising a classical estrogen response element.Preferred AP1 sites can be isolated from metalloprotease genes.Preferred classical estrogen response elements can be isolated from theXenopus vitellogenin A2 gene.

The methods typically use cells comprising an estrogen receptor and apromoter comprising an AP1 site which regulates expression of a reportergene. The cells are then contacted with the test compound and theexpression of the reporter gene is detected. The methods areconveniently used for testing compounds known to be antiestrogens forthe ability to activate transcription through the AP1 site.

In other embodiments the assays are used to test environmental compoundsfor estrogenic activity. Environmental estrogens will typically benon-steroidal compounds, which are effective agonists of the estrogenreceptor. Such compounds may be tested for their ability to activatetranscription through the classical estrogen response element or throughthe AP1 site. For this purpose, cells which express mutant estrogenreceptors are conveniently used. Cells expressing mutant estrogenreceptors with lower activity are useful in decreasing backgroundtranscription. A preferred cell is the ERC1 cell line.

Cells derived from a source other than breast tissue are generallypreferred for measuring activation mediated by the AP1 site. Forexample, uterine cells such as Ishikawa cells can be used. The reportergenes used to detect an estrogen response include genes encodingbeta-galactosidase and bacterial chloramphenicol acetyl transferase. Thepromoters used may be those which naturally comprise AP1 or estrogenreceptor elements or the promoters may be genetically engineered tocomprise those elements.

In some embodiments a single cell may comprise two promoters, each witheither the AP1 or the classical estrogen response element. In theseembodiments, two different reporter genes are operably linked to the twopromoters. In these assays the ability of test compound to induce orinhibit both the indirect and classical pathways can be determined.

When the methods are used to identify estrogen antagonists, the testcompounds are contacted with the cells and a compound known to mediatean indirect estrogen response. The ability to inhibit the response isdetermined by detecting the expression of the reporter gene. Compoundsknown to mediate an indirect estrogen response include tamoxifen andestrogen at half maximal concentrations. The compounds can also betested for the ability to induce or block the classical estrogenpathway, as well.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show estrogen stimulation mediated by an AP1 site inERC1 cells (FIG. 1A) and F9 cells (FIG. 1B). Typical results of CATassays, normalized for transfection efficiency, following a singletransfection are shown opposite. Each point is the mean value oftriplicate assays, with standard errors. CAT activities from cellsmaintained in the absence of hormone are shown as white bars, those inthe presence of a saturating concentration (100 nM) of estradiol asblack bars.

FIGS. 2A-2C show antiestrogen stimulation of expression in an intactAP-1 Site, but not at classical EREs. FIG. 2A shows CAT assays of HeLacells transfected with the indicated reporter genes and 3 μg of human ERexpression vector. Representations, at left, show the human collagenasepromoter (shaded) and the consensus AP-1 site. CAT activities were fromcells maintained in the absence of hormone or saturating concentrationsof ICI 164,384 (ICI, 1 μM), tamoxifen (5 μM) or estradiol (100 nM). CATactivity is normalized to a transfection control with the actin promoterdriving expression of βHCG. Single representative experiments are shown,error bars represent standard deviation of triplicate hormonetreatments. FIG. 2B shows CAT assays of HeLa cells transfected withreporter genes consisting of sequences overlapping the collagenase AP-1site (-73 to -52) upstream of the herpes simplex virus TK promoter (from-109 to +45 relative to the start site of transcription) or the nativeTK promoter alone. FIG. 2C shows CAT assays of HeLa cells transfectedwith reporter genes containing classical EREs.

FIGS. 3A and 3B show that antiestrogen agonism at AP-1 sites requiresER. FIG. 3A shows dose dependence of estrogen and antiestrogen inductionof coll73-LUC in HeLa cells relative to input ER expression vector,normalized to constant input DNA with blank expression vector SG5. Theluciferase assays were normalized to actin-HCG and were expressedrelative to values that were obtained with the collagenase promoter inthe absence of expression vector and hormone. A single representativeexperiment with triplicate points is shown. Error bars representstandard deviations. FIG. 3B shows concentration dependence of estrogenand antiestrogen induction of coll73-LUC in HeLa cells. HeLa cells weretransfected with 5 μg of ER expression vector and the collagenasepromoter active upon a luciferase reporter gene. The cells were exposedto a range of concentrations of ligand. Error bars represent standarddeviation of triplicate points.

FIG. 4A and 4B show that tamoxifen is an agonist in endometrial Cellsbut not in breast cells. FIG. 4A shows the response of the transfectedcoll73-CAT reporter in Ishikawa cells treated with estrogen orantiestrogen. Left panel, coll73-CAT response with endogenous ERcontrasted with the response of an ERE regulated reporter,ERE-coll60CAT. Right panel, coll73-LUC response with 3 μg co-transfectedexpression vector for human ER. Averages of three individual experimentsare shown. FIG. 4B shows activity of the collagenase promoter in breastcell lines with endogenous ER. Left panel shows response of thecollagenase promoter driving CAT in MCF7 cells (average of fourexperiments). Right panel shows activity of the coil promoter drivingluciferase expression in ZR75 cells, either without or with 300 ng ofhuman ER expression vector. A single representative experiment withtriplicate hormone treatments is shown.

FIGS. 5A-C show that hormone response at the AP-1 site requires AP-1proteins. FIG. 5A shows potentiation of hormone responses in HeLa cellsby Jun and Fos. Relative luciferase activities, normalized to HCGproduction, and calculated relative to collagenase expression in theabsence of ER and hormones are presented. The errors represent standarddeviations of three separate experiments. FIG. 5B shows effects of ERwith and without transfected Jun and Fos on hormone induction of thecollagenase promoter in F9 cells. Averages of five or six individualtransfections are shown. FIG. 5C shows response of the collagenasepromoter in F9 cells to increasing amounts of Jun, Fos, or theircombination, in the absence of ER.

FIGS. 6A-C show that the DNA binding domain of ER is required fortamoxifen induction at an AP-1 site, but not required for estrogeninduction. Reporters regulated by an AP-1 site (left panels), or an ERE(right panels) were introduced into Hela (FIG. 6A), CHO (FIG. 6B), orMDA453 cells (FIG. 6C), with 5 μg, 100 ng and 1 μg respectively of eachexpression vector for the ER derivative whose structure is indicated.The DNA binding domain is indicated with the striped box, the ligandbinding domain (AF2) and the amino terminal (AFI) activation functionsare marked. Results are presented for coll73-LUC in HeLa and MDA453cells and coll73-CAT in CHO cells. CAT and luciferase activities arecalculated relative to those obtained with coll73-LUC or coll73-CAT withSG5 blank expression vector in the absence of hormones.

FIGS. 7A and 7B show that fusing an exogenous transactivation functionto the ER increases activation at AP-1 sites. A luciferase reporterregulated by an AP-1 site (left panels) and a CAT reporter regulated byan ERE (right panels) were introduced into Hela (FIG. 7A), CHO (FIG. 7B)with expression vector for the ER derivative whose structure isindicated. CAT or Luciferase activities, normalized to HCG productionare shown. Activator plasmids, are shown schematically at the left ofthe figure. The VP16 transactivation domain is represented as an oval.The GALA DNA binding domain is marked.

FIGS. 8A and 8B show that fusing an exogenous transactivation functionto an ER derivative without the ligand binding domain potentiates geneexpression mediated by an ERE but not by an AP-1 site. A luciferasereporter regulated by an AP-1 site (left panels) and a CAT reporterregulated by an ERE (right panels) were introduced into HeLa cells withexpression vector for ER derivatives. CAT or Luciferase activities,normalized to HCG production, are shown.

FIG. 9 shows a library of hydroxystilbene derivatives tested in theassays of the invention.

FIG. 10 shows estrogenic activity of series 4 compounds illustrated inFIG. 9 as well as inhibition of activity by treatment with ICI.

FIG. 11 shows dose response curves for series 4 compounds.

FIG. 12 shows results of ER binding/competition assays for series 4compounds.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention provides an efficient way to screen large numbersof test compounds for those which have desirable properties for eitherthe treatment or the prevention of various cancers (e.g. breast cancer,ovarian cancer, endometrial cancer) and other diseases (e.g.endometriosis) mediated by estrogen. The invention thus provides methodsof screening for novel types of antiestrogen compounds that block theindirect estrogen response and/or block estrogen action at classicalestrogen response elements. As used herein an antiestrogen is a compoundthat substantially inhibits estrogen activity as measured in a standardassay for estrogenic activity, for example, cellular assays as describedin Webb et al. Mol. Endocrinol., 6:157-167 (1993).

The invention also allows for screening of test compounds for estrogenicactivity. The assays are particularly useful for screening environmentalcompounds suspected of having estrogenic activity, referred to here asxenoestrogens. Xenoestrogens are defined here to include any compoundhaving estrogenic activity in the assays described herein, which isderived from a source outside the human body. Environmental compounds asused herein can be derived from a wide variety of sources includingplants, soil, water, foods. They also include synthetic compounds suchas chlorinated organics, polycyclic aromatic hydrocarbons, herbicides,pesticides, pharmaceuticals and the like.

In animals and in man the balance between stimulatory and inhibitoryactivities of antiestrogens such as tamoxifen varies widely depending onthe organ, cell or specific protein measured as an indicator ofestrogenic activity. This variety of effects is difficult to reconcilewith the model of antagonism of estrogen receptor (ER) activity atclassical estrogen receptor elements (EREs) as described in Beato, M.Cell, 56: 335-344 (1989) and Klein-Hitpass, et al., Nucleic Acids Res.,16:647-663 (1988).

The present invention relies, in part, on the discovery that ERs mayactivate transcription by interaction with another response element, theAP i binding site, instead of binding to EREs. This AP1 mediatedpathway, referred to here as the indirect estrogen response, may accountfor much of the agonistic properties of tamoxifen and other putativeantiestrogens. A general description of the AP1 site is found in Angel &Kann, Biochem. Biophys. Acta., 1072:129-157 (1991) and Angel, et al.,Cell, 49: 729-739 (1987).

In the methods of the invention, both the classical estrogen responseelements and the indirect estrogen response may be used to provide ascreening system that detects both estrogen antagonistic and agonisticactivity. When testing an environmental compound for estrogenicactivity, the methods typically comprise cultured cells that producehigh levels of the human estrogen receptor. Such cells include MCF-7cells (ATCC No. HTB 22), MDA453 cells (ATCC No. HTB 131), ZR-75-1 cells(ATCC No. CRL 1500) or ERC1 cells described in Kushner et al., Mol.Endocrinol., 4:1465-1473 (1990). ERC2 and ERC3 cells as described byWebb, et al. Mol. Endocrinol., 6:157-167 (1993).

Cells expressing mutant estrogen receptors with decreased sensitivityfor estrogenic compounds are preferred for testing environmentalcompounds. Cells expressing the wild type receptor (e.g., MCF7 cells)have high background levels of transcription even in the absence ofhormone. Transcription induced by weakly active environmental compoundsmay be masked in these cells. Thus, preferred cells for this purposeinclude cells which over-express mutant estrogen receptors, such as theERC cells noted above.

When testing an antiestrogen compound's ability to activatetranscription through the AP1 mediated pathway, the source of the cellsused in the assay can influence the results. In particular, evidenceprovided below indicates that agonistic activity of an antiestrogen(e.g., tamoxifen) is usually strong in cells in which its agonisticactivity at an ERE is weak, and weak in cells in which its agonisticactivity at an ERE is strong. As shown below, antiestrogens show littleor no agonistic activity through the AP1 pathway in breast cancer celllines. Thus, when testing antiestrogens for agonistic activity in theAP1 pathway, cells other than breast cancer cell lines are usually used.For instance, cells of uterine origin such as cervical cells (e.g., HeLacells) or endometrial cells (e.g., Ishikawa cells) can be used. Theinvention is not limited to practice in mammalian cells and may bepracticed, for example in yeast and insect cells, transfected with theappropriate genes and recombinant constructs.

The cells may be modified to provide truncated or chimeric estrogenreceptors as described in Berry, et al., E. M. B. O. J., 9:2811-2818(1990). These modifications may result in increased estrogen affinityand increased sensitivity of the assay.

In addition, these cells are transfected with reporter genes in which aresponse element (either the AP1 site or ERE) regulates expression of areporter gene. Typically, two different reporter genes are used. Onegene reports transcription induced by the classical estrogen responsesystem, while the other gene reports transcription induced by theindirect estrogen response. The two reporter genes and response elementsare typically placed in separate cells, but the methods can also be usedwith both constructs in the same cell.

The reporter gene for the classical estrogen response system contains anestrogen response element (ERE) upstream of the target promoter andcapable of regulating that promoter. In a preferred embodiment the EREmay be the consensus estrogen response element AGGTCACAGTGACCT (SEQ IDNO: 1) from the Xenopus vitellogenin A2 gene.

The particular ERE used in the cells is not a critical aspect of theinvention and the present invention is not limited to the use of thisERE. Other EREs known to one of skill in the art can also be used. Forinstance, other sources of naturally occurring EREs include the B2 gene,the chicken ovalbumin gene, and the PS2 gene. Alternatively,non-naturally occurring EREs may be inserted into particular promoters.The consensus ERE from the Xenopus vitellogenin A2 gene is widely usedfor this purpose, but other EREs may be used as well.

The reporter gene for the indirect estrogen response pathway contains anAP1 site upstream of the target promoter and capable of regulating thatpromoter. The AP1 site is a sites that are bound by AP1 (the Jun and Fosproteins) or other members of that protein family. In a preferredembodiment, the consensus AP1 site is TGA(C/G)TCA.

One of skill would recognize that the particular AP1 site used is not acritical aspect of the invention. Any sequence capable of being bound byAP1 or members of that family and regulating a promoter is suitable.This would include promoters which encompass a naturally occurring AP1site. Typical promoters include, but are not restricted tometalloprotease genes such as stromelysin, gelatinase, matrilysin, andthe human collagenase gene.

Alternatively promoters may be constructed which contain a non-naturallyoccurring AP1 or related binding site. This facilitates the creation ofreporter gene systems that are not typically found under the control ofAP1. In addition, promoters may be constructed which contain multiplecopies of the AP1 site thereby increasing the sensitivity or possiblymodulating the response the reporter gene system.

The present invention is not limited to a particular reporter gene. Anygene that expresses an easily assayable product will provide a suitableindicator for the present assay. Suitable reporter genes are well knownto those of skill in the art. They include, for example, bacterialchloramphenicol acetyl transferase (CAT), beta-galactosidase, orluciferase.

One of skill will recognize that various recombinant constructscomprising the AP1 site can be used in combination with any cell or linewhich expresses a estrogen receptor.

To screen a number of compounds for antiestrogen action, cells with highlevel expression of human estrogen receptors and harboring either orboth response elements and reporter genes are exposed to doses ofestrogen which give half maximal induction or less. In each ease thiswill result in induction of several to hundreds of fold depending on thelevels of estrogen receptor and the particular details of the reporterconstruction. This will be reflected in increases of the reporter geneproduct, such as the CAT gene product which may be quantitated byenzymatic assay. The cells can be exposed to estrogens either growing inseparate wells of a multi-well culture dish or for colorometic assay ina semi-solid nutrient matrix. The antiestrogenic compounds to be testedare added to the culture dish wells or to small wells made in thesemi-solid matrix and the effect on the estrogen induction is assayed.An antiestrogen compound will reduce or abolish the estrogen inducedincrease in reporter gene activity. A hypothetical pure antiestrogenwill block estrogen action with both types of reporter genes and willhave no ability to induce the reporter genes in the absence of estrogen.A mixed estrogen antagonist-agonist, will show some ability to inducethe reporter genes, especially the reporter genes linked to AP1 site.

In other embodiments, environmental compounds suspected of havingestrogenic activity are contacted with cells with high level expressionof human estrogen receptors and harboring either or both responseelements and reporter genes as described above. Those compounds withestrogenic activity will result in induction of several to hundreds offold depending on the levels of estrogen receptor and the particulardetails of the reporter construction. Quantification of the activity byenzymatic assay can be carried as described above.

An assay for detection of xenoestrogens can be conveniently provided inkit format. Such a kit includes growth media, estrogen standards, andcells comprising the appropriate recombinant constructs. The kits mayalso include reagents suitable for detecting the product of the reporter.gene, and the like.

Antiestrogens which block the indirect pathway can be used to supplementtamoxifen or other antiestrogens in the treatment or prevention ofbreast cancer and other diseases mediated by estrogen. These compoundsfunction to eliminate estrogenic agonistic activity of antiestrogens.Second they may have uses by themselves. In particular, it may beadvantageous to block some estrogen mediated pathological effects atindirect estrogen response elements while leaving the direct pathwayactive. Compounds that block the indirect pathway are useful ascomponents of combined oral contraceptives (COC) containing estrogensand progestins. A triple COC, containing estrogens, progestins, and ablocking compound would allow estrogen, either in the formulation orendogenous to act at the classical response elements, but would blockaction at the indirect response elements. Thus, a triple COC functionsas current COCs to prevent pregnancy, but may also provide protectiveeffects against breast cancer.

Typically, the reporter gene linked to the AP1 site is activated, notwith estrogen, but with an excess (10 times the Kd) of tamoxifen,4-hydroxy tamoxifen, or other antiestrogen. A library of compounds issearched for candidates that block or reduce reporter gene activation bythe antiestrogen. The candidates are then tested with cells containingthe reporter gene for the classical pathway to confirm that they do notinterfere with estrogen activation at an ERE.

Antiestrogen or estrogen compounds identified in the assays of theinvention can be used in standard pharmaceutical compositions for thetreatment of cancer, as components of oral contraceptives, or any otherapplication in which the modulation of estrogen activity is desired. Thepharmaceutical compositions can be prepared and administered usingmethods well known in the art. The pharmaceutical compositions aregenerally intended for parenteral, topical, oral or local administrationfor prophylactic and/or therapeutic treatment. The pharmaceuticalcompositions can be administered in a variety of unit dosage formsdepending upon the method of administration. For example, unit dosageforms suitable for oral administration include powder, tablets, pills,and capsules.

Suitable pharmaceutical formulations for use in the present inventionare found in Remington's Pharmaceutical Sciences, Mack PublishingCompany, Philadelphia, Pa., 17th ed. (1985). A variety of pharmaceuticalcompositions comprising compounds of the present invention andpharmaceutically effective carriers can be prepared.

The following examples are offered by way of illustration, not by way ofimitation.

EXAMPLE 1

MATERIALS AND METHODS

Plasmid Construction

All reporter genes described below have been modified by digestion withEcoO109 and Ndel to remove an AP-1 site in the backbone of pUC. Thus,Coll73 and Coll60 are formerly ΔColl73 and ΔColl60 (Lopez et al. Mol.Cell. Biol. 13:3042-930 (1993)). Coll73-LUC was constructed by cloning aBamHI/PvuII fragment, that spanned the luciferase transcription unit,from pMG3 into coll73, which had been digested with BamHI and Sinai toremove the CAT transcription unit. EREcoll60 and EREcoll73 was preparedby ligation of a consensus ERE (AGGTCACAGTGACCT SEQ ID NO: 1), into theHindIII site upstream of coll60 and coll73, respectively. All otherreporter genes have been previously described (Webb et al. Mol.Endocrinol. 6:157-16725 (1992); and Lopez et al., supra).

Expression vectors for ER and ER mutants (Kumar et al. Cell 51:941-51(1987)), VP16-ER (Elliston et al. J Biol Chem 265:11517-21 (1990)),c-jun (Turner et al. Science 243:1689-94 (1989)) and c-fos (Sassone etal. Cell 54:553-60 (1988)) have been described. For this study, all ERcDNAs were cloned into the EcoRI site of the SG5 expression vector(Green et al. Nucleic Acids Res 16:369 (1988)).

The VP16ER cDNA was also cloned into SG5, to form the vector VER, in twosteps. The expression vector Vp16ER1-422 (Elliston et al., supra) wasdigested with SstI, repaired with T4 polymerase, and ligated to ECORIlinkers. The resulting EcoRI fragment was sub-cloned at the equivalentsite of SG5 to generate the vector VER1-422. This was digested tocompletion with HindIII and BgIII and the equivalent (HindIII/BamHI)fragment from HEO was replaced at this location. VERΔDBD was constructedby substituting a Not1/BgIII fragment from HE11 (cloned in SG5) into VERdigested with the same enzymes.

The GST wild type ER fusion gene (GST-HEGO) was constructed by ligationof the EcoRI fragment from pSG5-HEGO, spanning the ER cDNA, intopGEX5X-1, one of the vectors of the pGEX series (Pharmacia Biotech Inc.,Piscataway, N.J., USA). GST-hELBD was constructed in two steps. An Xbafragment from HE19G was inserted into the equivalent position of XbaIdigested SG5-HE14, which spans the ER LBD (Kumar et al. ). Then, anEcoR1 fragment spanning this ER cDNA was cloned into pGEX-3X. To prepareGST-hEN185 an EcoRI/KpnI fragment spanning the ER amino terminus wasobtained from the vector EGE, repaired and cloned into pGEX-5X-1digested with SmaI and EcoRI.

Tissue Culture and Transfections

Cells were maintained and transfected as previously described in Webb etal., supra. Hormones were added two hours after plating in the followingconcentrations, estradiol 100 nM, ICI 164,384 1 μM, tamoxifen 5 μM, toensure saturation of the response. F9 cells were seeded at 30%confluence upon 1.5 cm dishes and transfected overnight by calciumphosphate coprecipitation with 5 μg reporter gene, 1 μg actin β-HCG, and1 μg of HEO, 300 ng each of c-jun and c-fos expression vectors. Thecells were glycerol shocked and refed in growth medium containinghormone or ethanolic vehicle. In transient transfections, optimal mountsof HEO were employed and were as follows: HeLa (5 μg); NIH3T3 (1 μg);HepG2 (1 μg); SHM (300 ng); SY5Y (300 ng); CEF (100 ng); CV-1 (3 μg);MDA453 (3 μg); CHO (100 ng) and F9 (1 μg).

CAT assays were carried out as described in Webb et al., except that thecells were harvested one to two days after transfection instead ofthree. CAT activities were defined as the increase in cpm per hour atroom temperature (corrected for background) per 100 μl of cell extract,normalized to production of 100 standard units of βHCG, from aco-transfected reporter gene, actin-HCG. Luciferase assays wereperformed as described by Brasier et al. Methods Enzymol 216:386-97(1992)) on similar extracts that were used for CAT assays. Light unitswere defined as the luciferase activity per 100 μl of cell extract per100 standard units of βHCG. Relative luciferase activities werecalculated with respect to the results that were obtained in the absenceof ER and hormone, which was set at 1 unit. For the data presented inTable I triplicate points were determined. Standard deviations were lessthan 20%.

GST Fusion Protein Binding Assay and in vitro Translation

Procedures were carried out as described by Lopez et al., supra.Briefly, fusions of GST to various domains of the human ER were preparedas follows. Bacteria expressing the fusion proteins were resuspended inbuffer IPAB-80 (20 mM REPES, 80 mM KCl, 6 mM MgC!2, 10% Glycerol, 1 mMDTT, 1 mM ATP, 0.2 mM PMSF and protease inhibitors; pH 7.9), sonicatedmildly, and the debris was pelleted at 12,000 rpm for 1 hr in an ss34rotor. The supernatant was incubated for 2 hrs. with 500 μL ofglutathione sepharose 4B beads that were previously washed with 5volumes of PBS 0.2% Triton X-100 and equilibrated with 5 volumes of IPAB80. GST-fusion proteins beads were then washed with 5 volumes of PBS0.05% Nonidet P-40 and resuspended in 1 ml of IPAB-80 for storage at 4 Cuntil use. All the above procedures were done in a cold room at 4 ° C.

Assays of GST-ER fusions were carded out in 100 μL volume that contained40 μL of bead suspension (equivalent to 10 μL of compact beads volume)and 1 to 2 μL of 35S in vitro translated c-jun or c-fos in IPAB-80 2.5%non fat milk and incubated for 1.5 hr at 4° C. Beads were washed 5 to 6times with IPAB-80 containing 0.05% Nonidet P-40. Input labelledproteins, proteins bound to GST, GST-hER and other ER fusion beads werethen subjected to SDS polyacrylamide gel electrophoresis (PAGE) in 10%acrylamide and then to autoradiography.

RESULTS

Antiestrogens activate transcription through the AP1 site.

The human collagenase gene, like other matrix metalloproteases, respondsto AP1. The promoter from this gene contains a consensus AP-1 sitelocated between -60 and -73 base pairs from the start of transcription.Angel, et al., Mol. Cell. Biol., 7: 2256-2266 (1987). To test whether anAP1 site could confer estrogen response the collagenase promoter wasfused to the bacterial CAT gene (Δcoll73) and transfected into ChineseHamster Ovary cells that over-express ER (ERC1) Kushner, et al. Mol.Endocrinol. 4:1465-1473 (1990).

Estradiol stimulated Δcoll73 ten fold (FIG. 1A), whereas a similarreporter in which the AP1 site had been removed (Δcoll60), gave reducedbasal activity and no estrogen response. Substitution of a classical ERE(Klein-Hitpass, et al., Nucl. Acids Res. 16:647-663 (1988)) for the AP1site (EREΔcoll60), restored estrogen response, but not the elevatedbasal activity.

In F9 cells, which lack endogenous AP1 activity (Chiu, et al. Cell, 54:541-551 (1988)) Δcoll73 failed to respond to estrogen in the presence oftransiently transfected ER (FIG. 1B). Estrogen activation could berestored by cotransfecting expression vectors for AP1 proteins c-Jun andc-fos. Sassone-Corsi, P. et al., Cell, 54: 553-560 (1988). Turner &Tjian, Science 243:1689-1694 (1989). In parallel, EREΔcoll60 wasestrogen responsive, even in the absence of AP1, and Δcoll60 andremained unaffected by estrogen (data not shown). Estrogen induction ofthe collagenase promoter therefore required both the AP1 site and AP1proteins.

To further examine the effects of antiestrogens on the AP-1 directedpathway, reporter genes derived from the human collagenase promoter weretransfected into HeLa cells. Both estrogen and antiestrogens activatedthe collagenase promoter in the presence of transiently expressed humanER (coll517, FIG. 2A). In these cells tamoxifen was more potent anactivator than estrogen. This pattern was retained with coll73, but waslost with coll60 or was inactivated by point mutations (coll517mAP1).

When the collagenase AP-1 site was placed upstream of the herpes virustk promoter both tamoxifen and ICI were able to activate transcription,although this response was not as robust as with the native collagenasepromoter (FIG. 1B). These results indicate that antiestrogens areagonists at the collagenase promoter and a heterologous promoter linkedto AP-1. Thus, the AP-1 site is required for this activity.

The activity of antiestrogens in the AP-1 pathway was also compared withtheir activity in the classical pathway. Direct substitution of an EREfor the collagenase AP-1 site restored estrogen response to the corecollagenase promoter, but not antiestrogen response or the basalactivity associated with the AP-1 site. A promoter with both an ERE andan AP-1 site (ERE-coll73 FIG. 2C) gave a large estrogen response, butretained some response to antiestrogens. Another control reporter, inwhich the tk promoter was regulated by a classical ERE (ERE-tk FIG. 2C)was also activated by estrogen, but not by antiestrogens. Thus, aclassical ERE cannot substitute for the AP- 1 site, indicating that theAP-1 site has a unique function in activation by antiestrogens.

To determine whether ER was required for antiestrogen agonism theresponse of the collagenase promoter, upstream of the luciferase gene(coll73-LUC), to transfection of increasing mounts of ER into HeLa cellswas examined (FIG. 3A). Estrogen and antiestrogen responses were notseen in the absence of ER, and increased as a function of the mount oftransfected ER expression vector. Tamoxifen responses were more potentthan estrogen responses at every level of receptor.

The effect of increasing doses of each ligand was also examined. FIG. 3Bshows that the half maximal dose for ICI is about 10 times, andtamoxifen 100 times, that for estrogen. This is consistent with theknown binding affinities of these compounds to the estrogen binding siteon the receptor and suggests that they are stimulating transcriptionthrough that site. Similar half maximal doses were obtained for bothestrogen and the weak tamoxifen responses that were seen at a classicalERE (dam not shown).

In summary, indirect estrogen response is widely active, andantiestrogens are agonists of this pathway. It is possible that any ofthe well described agonist effects of tamoxifen reflects indirectestrogen response. Antiestrogens would have estrogenic activity oncritical AP1 regulated target genes, hence growth and differentiatedresponse, in cells in which ER and AP1 proteins could interact. Changesin AP1 during tumor progression could be particularly significant, andshould be considered in models of antiestrogen resistance in breastcancer. Parker, et al. Cancer Surveys, 14, Growth Regulation by NuclearHormone Receptors. Cold Spring Harbor Laboratory Press (1992).

Antiestrogens Are Agonists of the AP-1 Pathway in Many Cell Types

The data above show that antiestrogens are agonists at the AP-1 drivencollagenase promoter, but not at classical EREs, in HeLa and othercells. To test whether this pattern was widespread, the effect ofestrogen and antiestrogens on the expression of reporter genes driven byeither the native collagenase promoter, or a similar promoter in whichthe AP-1 site was replaced by a classical ERE, was tested in a range ofcell lines. In each case, the cells were transfected with differentamounts of the human ER expression vector HEO to determine the optimalresponse.

Table I shows that both estrogen and antiestrogens activated thecollagenase promoter in most cell types. This response occurred withcell lines representative of different tissue types including cervix,liver, myometrium, neuroblastoma, kidney and ovary. In most casestamoxifen was as potent, or more potent, than estrogen. Only F9 cells,which have low levels of endogenous AP-1 activity, were not activated byany ligand.

In the same range of cell types both antiestrogens displayed littleactivity at classical EREs (data not shown). ICI consistently behaved asa pure antagonist of ER action at an ERE. In HeLa cells, and most othercases, tamoxifen inductions of ERE-coll60 activity remained at less than3% of those obtained with estrogen. Significant (30% of estrogen)tamoxifen inductions at classical EREs were obtained in CEF cells andCV-1 cells and MDA453 cells. In these latter cells tamoxifen action atthe AP-1 site was relatively weak (Table I). Thus, tamoxifen activity atan AP-1 site may be strong in cells at which its activity at an ERE isweak (HeLa), and weak at an AP-1 site in cells at which its activity atan ERE is strong (MDA453).

In conclusion, antiestrogen agonist effects occur at AP-1 sites in cellsof diverse origin. These effects show little correlation with theactivity of tamoxifen at classical EREs.

Antiestrogens are Agonists of the AP-1 Pathway In Endometrial CellLines, But Not in Breast Cells

The data in Table I show that, in most cell types, tamoxifen was atleast as potent as estrogen in inducing the collagenase promoter. In onecell line, MDA453 breast cancer cells, the AP-1 driven collagenasepromoter was activated efficiently by estrogen but not by tamoxifen.Similarly, in Chinese hamster ovary (CHO) cells, estrogen inductionsroutinely exceeded antiestrogen inductions. This suggests that tamoxifenaction at the collagenase promoter might have a cell specific component.

To further explore this phenomenon, and to test whether similar hormoneeffects could be detected at physiological levels of ER, the expressionof the collagenase promoter in cells that express endogenous ER wasexamined. Ishikawa cells, an endometrial cell line that is believed torepresent a model of tamoxifen agonism on the uterus (described byHolinka, et al., J. Steroid Biochem., 25:781-786 (1986)), and two breastcancer cell lines, MCF-7 and ZR-75-1, both of which are known to respondto estrogen but not tamoxifen, were used.

In Ishikawa cells (FIG. 4A) the collagenase promoter was activated byestrogen and tamoxifen, but the ICI compound was usually inactive in thepresence of endogenous receptor. This parallels the reported potency oftamoxifen and ICI on cell growth and induction of progesterone receptorsin these cells. When receptor levels were raised by transfection,tamoxifen and estrogen inductions became larger and ICI inductionsbecame detectable. In contrast, neither ICI nor tamoxifen activatedexpression of ERE-coll60, whereas estrogen induced expression of thisreporter gene tenfold. Tamoxifen also failed to activate several othergenes that contain simple classical EREs in Ishikawa cells (data notshown).

In MCF-7 cells, estrogen, but not tamoxifen, activated the collagenasepromoter (FIG. 4B). The same pattern occurred in ZR-75-1, and could beseen more clearly when extra receptors were supplied by transfection.Again, this resembles the results that were obtained in MDA453 cells(Table I), and parallels the reported absence of tamoxifen effects oncell proliferation and gene expression in breast cancer cell lines.

In conclusion, tamoxifen activates the collagenase promoter in cellswith physiological levels of ER, and the response shows tissuerestrictions. Tamoxifen activity occurs in endometrial cells, but not inbreast cells, and thus parallels the known tissue specificity oftamoxifen agonism.

                  TABLE I                                                         ______________________________________                                                         Coll73 Luciferase Activity (*1)                                                     No                                                     Cell Line                                                                             Origin         Hormone   ICI  Tam  E2                                 ______________________________________                                        HELA    CERVIX         1.0       3.7  7.4  3.4                                NIH 3T3 FIBROBLAST     1.0       3.0  3.1  3.3                                HEP G2  LIVER          1.0       5.2  6.5  4.3                                SHM     MYOMETRIUM     1.0       1.9  2.2  2.0                                SY5Y    NEUROBLASTOMA  1.0       2.3  2.1  2.1                                CEF     FIBROBLAST     1.0       3.2  2.2  2.5                                CV-1    KIDNEY         1.0       3.1  5.3  2.2                                MDA453  BREAST         1.0       1.1  2.   8.4                                CHO     OVARY          1.0       1.8  2.2  3.3                                F9 (*2) TERATOCARCINOMA                                                                              3.2       2.4  1.1  1.4                                ______________________________________                                         (*1) Activities were determined in triplicate transfections. Activities       were normalized to an actinHCG internal control and expressed relative to     values obtained from the collagenase promoter in cells that were not          transfected with ER or treated with hormone (see Materials and Methods).      Standard deviations (not shown) were less than 20%.                           (*2) Unliganded ER increased the basal activity of the collagenase            promoter.                                                                

AP-1 Proteins Are Required for ER Action at the Collagenase Promoter

To test whether AP-1 proteins, as well as their cognate binding site,were required for the AP-1 pathway, we examined whether Jun and Fosoverexpression affected the hormone response of the collagenasepromoter. The Examples above establish that antiestrogens and estrogensactivate the collagenase promoter in HeLa cells in the presence of ER(see, e.g., Table I). FIG. 5A shows that these inductions are markedlyincreased by the presence of transfected AP-1, especially in thepresence of Jun or Jun/Fos. This suggests that Jun homodimers or Jun/Fosheterodimers occupying the AP-1 site contribute to the ability of ER toactivate description in the AP-1 directed pathway.

To confirm that AP-1 proteins were absolutely required for the AP-1directed ER pathway, we turned to F9 cells, which have only low levelsof endogenous AP-1 activity. Transfection of an expression vector forestrogen receptor into these cells did not support hormone activation ofthe collagenase promoter (Table D, whereas it gave strong estrogenactivation at an ERE (not shown). Co-transfection of ER with Jun/Fosrestored induction by both estrogen and antiestrogens in F9 cells,albeit at lower levels than that seen in HeLa cells. In addition therewas some activation by unliganded ER. Thus, the inability of F9 cells toallow a hormone response at the collagenase promoter can be overcomewith AP-1 supplied by transfection. We conclude that hormone effects atthe AP-1 site require AP-1 protein. However, the dramatic differencebetween the hormone response of HeLa and F9 cells when both are suppliedwith Jun and Fos indicates that other cell specific factors, in additionto AP-1 abundance, regulate the strength of the AP-1 directed ERpathway.

It is unlikely that ER dependent activation at AP-1 sites is due tochanges in the mount of AP-1. In these studies we determined the mountsof AP-1 required for optimal collagenase promoter activity in F9 cells.FIG. 5C shows that Jun, Fos, and a combination of both, increased basalactivity of the collagenase promoter (in the absence of ER) whichreached a maximum with 300 ng of expression vector. These mounts wereemployed in the co-transfections with ER (FIG. 5B). Thus, ER activationat AP-1 sites appears to increase the transcriptional efficiency of Junand Fos even when they are provided at optimal mounts.

ER Binds Jun But Not Fos in vitro

To test whether ER effects upon AP-1 might reflect direct biochemicalinteraction between the ER and AP-1 proteins, we examined whether theyspecifically interact in solution. An estrogen receptor protein fused toglutathione S-transferase (ER-GST), and attached to agarose beads,pelleted in vitro translated Jun from solution, whereas a control GSTprotein pelleted only background amounts of Jun. Similar bindingoccurred with the ER amino terminal domain, but not with the LBD.Neither the intact ER nor its isolated domains bound Fos. These resultsindicate that Jun, but not Fos, binds ER in vitro, and that a majortarget of Jun is the ER amino terminus.

Tamoxifen Activation at AP-1 Requires the ER DBD, Whereas EstrogenActivation Is DBD Independent In Some Cell Types.

We next examined which domains of the ER mediate hormone action. Weintroduced truncated derivatives of the ER into three different celltypes. We chose the HeLa, CHO and MDA453 lines as recipients because theER driven AP-1 pathway showed different properties in each cell. In HeLacells tamoxifen response predominated, in MDA453 cells estrogen responsepredominated, and CHO cells gave an intermediate phenotype (Table I). Weexamined the ability of each truncated ER to activate a reporter genedriven by the collagenase promoter with its AP-1 site (FIG. 6, leftside) or a reporter gene driven by control promoter with an ERE (FIG. 6,right side). Previous work has established that each of these variantERs is expressed at comparable levels from these vectors.

Deletion of the DNA binding domain (DBD) completely eliminated estrogenactivation at an ERE in all three cell types (HE11, FIG. 6). Deletion ofthe DNA binding domain also eliminated tamoxifen activation at AP-1sites, be it the substantial tamoxifen activation in HeLa and CHO cells,or the marginal mount in MDA cells. In contrast, removal of the DBD didnot abolish estrogen activation at the AP-1 site in any of the celllines. Indeed, estrogen activation at the AP-1 site in CHO cells wasequally strong with or without the ER DBD. This is consistent withprevious observations that estrogen response at AP-1 sites showsindependence of DNA binding in CEF. Thus, the requirement for the ER DBDvaries according to the ligand, it is required for tamoxifen inductionbut not estrogen induction. We suggest below (Discussion) that thedifferential requirements for the ER DBD may indicate the existence ofmore than one pathway of ER action at AP-1 sites.

The ER amino terminus also played an important role in tamoxifen andestrogen activation at the AP-1 site. Although deleting of the aminoterminus (HE19) did not eliminate activity upon the ERE regulatedreporter in all three cell types, this deletion abolished the strongtamoxifen-activation at the AP-1 site in HeLa cells and the weakertamoxifen activation in CHO and MDA453 cells. Deletion of the aminoterminus also markedly reduced estrogen activation at the AP-1 site inall three cell types.

A deletion of the ligand binding domain (HE15), leaving the aminoterminus and DBD intact, gave a constitutively active receptor that wasable to weakly activate at an ERE in all three cell lines. Thisreceptor, however, showed highly potent activity at the AP-1 site inHeLa cells, which correlated with the levels of activity obtained withthe tamoxifen liganded native ER. In contrast, HE15 was inactive inMDA453 cells and weak in CHO cells. Thus, the requirement of the ERamino terminus for AP-1 activation also shows cell type specificity, ina manner that correlates with the cells ability to support a tamoxifenresponse at the collagenase promoter. This again suggests thatactivation through AP-1 may occur through more than one mechanism.

ER Can Target an Exogenous Transactivation Domain to the CollagenasePromoter, Independently of the ER DBD

One possible mechanism for ER activation at AP-1 sites is that thereceptor might directly bind to the AP-1 complex at the promoter andfrom there influence transcription. A prediction of this model is thatER should be able to target heterologous transcriptional activationfunctions to an AP-1 regulated promoter.

In order to test this proposition, we examined the effects of linkingthe strong VP16 transcriptional activation domain to the amino terminusof the ER (V-ER). To monitor activity we used a luciferase reporter generegulated by an AP-1 site and CAT reporter gene driven by an otherwiseidentical promoter with an ERE. The V-ER chimeric receptor gave markedlyenhanced activation at an ERE in HeLa cells (FIG. 7A). It was activatedboth by estrogen and antiestrogens reflecting the ability of VP16 tooverride the need for AF-2 (see, Kumar et al. Cell 51:941-951 (1987)),and consistent with previous reports for this "super-receptor". Incontrast, the super-receptor had little effect at the AP-1 site in HeLa.Tamoxifen activation with the full length ER was hardly increased,although estrogen activation was modestly potentiated. We also tested aversion of the super-receptor in which the ER DBD was deleted (VERΔDBD).This receptor, as expected, failed to activate at an ERE. It was,however, more potent than an equivalent ER (HE11) that lacked the VP16activation function when tested at an AP-1 site.

To further explore this phenomenon we performed a series of similarexperiments in CHO cells (FIG. 7B), in which estrogen response at theAP-1 site was completely independent of the ER DBD (FIG. 6B). Onceagain, the V-ER chimera superactivated gene expression that was drivenby the ERE. In this case, however, V-ER also superactivated at the AP-1site. Although the superreceptor that lacked the DBD (VERΔDBD) remainedunable to activate transcription from an ERE, it was even more activethan V-ER at the AP-1 site. A control fusion of the VP16 domain to theyeast GAL4 DNA binding domain did not increase collagenase promotertranscription. Thus, the superactivation by VP16 in CHO cells isdependent upon sequences in the ER protein. These observations indicatethat super-receptors are super-activators at AP-1 sites in CHO cells ina DBD-independent manner. Similar results were also obtained with MDA453cells (data not shown). The contrast between the properties of thesuper-ER in HeLa and CHO cells further suggests that there may be morethan one pathway of activation at AP-1 sites.

VP16 Potentiates the Action of an ER Without an LBD at an ERE, But Notat an AP-1 Site.

The results described above suggest that addition of the VP16 activationfunction to native ER was unable to strongly potentiate tamoxifen actionat an AP-1 site in HeLa cells. We also observed that an ER lacking theLBD (HE15) was a potent constitutive activator of the AP-1 pathway inHeLa, and that this correlated with the ability of these cells tosupport a large tamoxifen response at the collagenase promoter. Todirectly test whether transcriptional activation functions were involvedin this pathway, we examined the effects of fusing the VP16 activationdomain to this receptor (V-ER302C, FIG. 8). FIG. 8B shows that thepresence of the VP16 domain greatly potentiated transcription from anERE, but failed entirely to potentiate transcription activation by ERfrom the AP-1 site (FIG. 8A). Indeed, the presence of the VP16 domainslightly decreased the activity of HE15 at the AP-1 site. Similarresults were also obtained in CHO cells (data not shown). Thus theactivation pathway of the LBD deleted receptor at AP-1 sites appears notto respond to exogenous transcriptional activation functions. We arguebelow that this suggests the existence of an ER pathway that activatestranscription from AP-1 sites independent of ER associatedtranscriptional activation functions.

EXAMPLE 2

Estrogenic Activity Screens

A library of hydroxystilbene derivatives as shown in FIG. 9 was screenedfor estrogenic activity in cell culture assays using a CAT reporter genelinked to a classical ERE in ERC 1 cells as described in Webb et al.,supra. After transient transfection with CAT reporter genes, eachhydroxystilbene was added to the cultured ERC1 cells. ER-regulatedresponse was compared either to treatment with 17β-estradiol as acalibration standard or to treatment with an ethanolic vehicle.Hydroxystilbene series 1, 2 and 3 showed no measurable estrogenicactivity data (data not shown), whereas series 4 compounds showed weakestrogenic activity (FIG. 10). Of the series 4 compounds 4A, 4E and 4Fwere found to provide the highest levels of estrogenic activity relativeto 17β-estradiol.

To examine whether the series 4 compounds induce estrogenic activitythrough the ER, we tested the ability of ICI164384 to inhibit the series4 estrogenic activity. The ICI164384 compound was found to inhibit theestrogenic activity of all the series 4 compounds (FIG. 3). As anegative control, CHO cells, which lack functioning ER, were transfectedwith the same estrogen-responsive reporter constructs and treated with17β-estradiol and the series 4 hydroxystilbenes. As expected, noestrogenic activity was seen with these cells.

Dose Response and ER Binding of Series 4

Dose response experiments were performed on the series 4 compounds overa concentration range of 0-100 μM (FIG. 11). For the most activecompounds, 4A, 4E, and 4F saturation is observed at 50 μM. The effectiveconcentration that provides 50% maximum activity (EC₅₀) ranges from -5μM to ˜15 μM for these three compounds. In vitro ER binding assays wereperformed on the three most active series 4 compounds to confirm thatthe estrogenic activity measured in the bioassay correlated with bindingaffinity for the ER. The inactive hydroxystilbene analog 3D was includedin the binding assay as a negative control. The ER-binding results forcompounds 4A, 4E and 4F are consistent with the estrogenic bioassay, aseach of the compounds show IC₅₀ values of 1-10 μM for ER binding (FIG.12). The two most active compounds in the bioassay, 4A and 4F, also showthe highest affinity (1 μM) for the ER, although this affinity isapproximately four orders of magnitude lower than that of 17β-estradiol.The analog 3D which showed no activity in the bioassay also shows nobinding affinity for the ER.

Additional experiments were performed to characterize further theestrogenic activity of the series 4 compounds identified in the initialscreen. Four of the compounds (4A, 4B, 4E and 4F) showed clear doseresponse profiles over a concentration range of 0-100 μM (FIG. 10). Thethree most active compounds, 4A, 4E and 4F, show maximum activity at 50μM and have EC₅₀ values for estrogen response in the range of 5-15 μM.Three lines of evidence suggest that the series 4 estrogenic response ismediated by direct binding of the hydroxystilbene to the estrogenbinding site of the ER. First, the fact that no estrogenic activity wasobserved in reporter gene-transfected CHO cells which lack a functionalER provides evidence that the response to the series 4 compounds wasER-mediated. Second, the observation that the response initiated by allthe series 4 compounds could be inhibited by ICI 164384, a potentsteroidal antiestrogen that competes with 17 β-estradiol for binding tothe ER, provides evidence that the hydroxystilbenes act through directbinding to the steroid binding site on the ER. Third, the bindingaffinity of the series 4 compounds to the ER was directly measured in acompetition binding assay with 17 β-estradiol and the measured IC₅₀values for the series 4 hydroxystilbenes correlate approximately withthe EC₅₀ values measured from the dose response bioassay.

Structure-activity relationships (SAR) in both the hydroxy-substitutedand distal aromatic rings of the hydroxystilbenes are evident from thevarying estrogenic activities of the library. Based on the observationthat activity was only seen in the series 4 compounds, it appears that apara orientation between the hydroxyl substituent and the stilbeneolefin is a requirement for ER-binding and activation. The paraorientation is sensitive to additional substitution as evidenced by thefact that no estrogenic activity was observed for the series 3(4-hydroxy-3-nitro) compounds; it is unclear whether steric orelectronic factors are responsible for the lack of activity in series 3.For the series 4 compounds, the three most active hydroxystilbenes beareither small fluorine substituents (4E, 4F) or no substitution (4A) inthe distal aromatic ring. This distal ring SAR is somewhat subtle: basedon the dose-response data, the 4'-Br substituted compound (4C) showsalmost no activity, whereas the 4'-F substituted compound (4F) is thesecond most active member of the series. Here again, it is unclearwhether steric effects, electronic effects, or a combination of both areresponsible for the variations in estrogenic activity.

Conclusion

The above results show that three of the hydroxystilbene analogspermeate cell membranes and trigger a dose-dependent estrogenic responsewith EC₅₀ values in the range of 5-15 μM. Results fromcompetition-response and ER-binding experiments with the antiestrogenIC1164384 and 17 β-estradiol provide evidence that the non-steroidalhydroxystilbene analogs elicit the estrogenic response through directinteraction with the steroid binding site of the estrogen receptor. Inaddition, structure-activity relationships for the hydroxystilbenepharmacophore are evident from the activity profile of the library. Suchinformation could prove useful for predicting potential estrogenicactivity of environmental pollutants and pharmaceuticals.

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AGGTCACAGTGACCT15                                                             __________________________________________________________________________

What is claimed is:
 1. A method for screening a test compound known tohave antiestrogenic activity for agonistic estrogenic activity mediatedthrough an indirect estrogen response, the method comprising:a)providing a cell comprising AP1 proteins, an estrogen receptor, and aconstruct comprising a promoter comprising an AP1 site which regulatesexpression of a reporter gene; b) contacting said cell with said testcompound known to have antiestrogenic activity; and c) detecting theexpression of said reporter gene wherein enhanced expression of saidreporter gene indicates that said test compound has agonistic estrogenicactivity mediated through an indirect estrogen response.
 2. The methodof claim 1, wherein the cell is an Ishikawa cell.
 3. The method of claim1, wherein the cell is genetically engineered to express the estrogenreceptor at higher levels than said cell without the geneticengineering.
 4. The method of claim 3, wherein said cell is an ERC1cell.
 5. The method of claim 1, wherein the promoter is geneticallyengineered to comprise an AP1 site.
 6. The method of claim 1, whereinthe cell is derived from uterine tissue.
 7. The method of claim 6,wherein the cell is a HeLa cell or an Ishikawa cell.
 8. The method ofclaim 1, wherein said antiestrogenic activity is determined by a methodcomprising the steps of:a) providing a second cell comprising anestrogen receptor and a construct comprising a promoter comprising astandard estrogen response element which regulates the expression of asecond reporter gene; b) contacting said second cell with said testcompound and a second compound known to have agonistic estrogenicactivity mediated through a direct estrogenic response; and c) detectingthe expression of said second reporter gene, wherein inhibition ofexpression of said second reporter gene produced by said compound knownto have agonistic estrogenic activity mediated through a directestrogenic response indicates that said test compound has antiestrogenicactivity.
 9. The method of claim 8, wherein the response element is fromthe Xenopus vitellogenin A2 gene.
 10. The method of claim 1, whereinsaid cell further comprises a construct comprising a promoter comprisinga standard estrogen response element which regulates expression of saidsecond reporter gene.
 11. The method of claim 10 wherein said standardestrogen response element Is from the Xenopus vitellogenin A2 gene. 12.A method for screening a test compound for the ability to inhibitagonistic estrogenic activity mediated through an indirect estrogenresponse, the method comprising:a) providing a cell comprising AP1proteins, an estrogen receptor, and a construct comprising a promotercomprising an AP1 site which regulates expression of a reporter gene; b)contacting said cell with said test compound and a compound known tohave agonistic estrogenic activity mediated through an indirect estrogenresponse; and c) detecting the expression of said reporter gene whereininhibition of enhanced expression of said reporter gene produced by saidcompound known to have agonistic estrogenic activity mediated through anindirect estrogen response indicates that said test compound inhibitsagonistic estrogenic activity mediated through an indirect estrogenresponse.
 13. The method of claim 12, wherein the compound known to haveagonistic estrogenic activity mediated through an indirect estrogenresponse is tamoxifen.
 14. The method of claim 12, wherein the cell isgenetically engineered to express the estrogen receptor at higher levelsthan said cell without the genetic engineering.
 15. The method of claim14, wherein said cell is an ERC1 cell.
 16. The method of claim 12,wherein the promoter is genetically engineered to comprise an AP1 site.17. A method for screening a test environmental compound for agonisticestrogenic activity mediated through an indirect estrogen response, themethod comprising:a) providing a cell comprising AP1 proteins, anestrogen receptor, and a construct comprising a promoter comprising anAP1 site which regulates expression of a reporter gene; b) contactingsaid cell with said test environmental compound, and c) detecting theexpression of said reporter gene wherein enhanced expression of saidreporter gene indicates that said test environmental compound hasagonistic estrogenic activity mediated through an indirect estrogenresponse.
 18. The method of claim 17, wherein said cell furthercomprises a construct comprising a promoter comprising a standardestrogen response element which regulates expression of a secondreporter gene.
 19. The method of claim 17, wherein the reporter gene isCAT.
 20. The method of claim 17, wherein the cell is geneticallyengineered to express the estrogen receptor at higher levels than saidcell without the genetic engineering.
 21. The method of claim 17,wherein the cell is an ERC1 cell.
 22. A method for screening a testcompound for the ability to activate transcription mediated through anindirect or direct estrogen response, the method comprising:a) providinga cell comprising AP1 proteins, an estrogen receptor, and a constructcomprising a promoter comprising an AP1 site which regulates expressionof a first reporter gene; b) contacting said cell with said testcompound; c) detecting the expression of said first reporter genewherein enhanced expression of said first reporter gene indicates thatsaid test compound has agonistic estrogenic activity mediated through anindirect estrogen response; d) providing a second cell comprising anestrogen receptor and a construct comprising a promoter comprising astandard estrogen response element which regulates expression of asecond reporter gene; e) contacting said second cell with said testcompound; and f) detecting the expression of said second reporter genewherein enhanced expression of said second reporter gene indicates thatsaid test compound has agonistic estrogen activity mediated through adirect estrogen response.
 23. The method of claim 22, wherein said firstcell is an Ishikawa cell.
 24. The method of claim 22, wherein said firstcell is genetically engineered to express estrogen receptors at a higherlevel than the same cell prior to said genetic engineering.
 25. Themethod of claim 22, wherein said first cell is derived from uterinetissue.
 26. The method of claim 25, wherein the cell is a HeLa cell oran Ishikawa cell.
 27. The method of claim 22, wherein said standardestrogen response element is from the Xenopus vitellogenin A2 gene.